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To validate serum neutrophil gelatinase-associated lipocalin (NGAL) as an early biomarker for acute kidney injury in critically ill children with septic shock.Observational cohort study.Fifteen North American pediatric intensive care units (PICUs).A total of 143 critically ill children with systemic inflammatory response syndrome (SIRS) or septic shock and 25 healthy controls.None.Serum NGAL was measured during the first 24 hrs of admission to the PICU. Acute kidney injury was defined as a blood urea nitrogen concentration >100 mg/dL, serum creatinine >2 mg/dL in the absence of preexisting renal disease, or the need for dialysis. There was a significant difference in serum NGAL between healthy children (median 80 ng/mL, interquartile ratio [IQR] 55.5–85.5 ng/mL), critically ill children with SIRS (median 107.5 ng/mL, IQR 89–178.5 ng/mL), and critically ill children with septic shock (median 302 ng/mL, IQR 151–570 ng/mL; p < .001). Acute kidney injury developed in 22 of 143 (15.4%) critically ill children. Serum NGAL was significantly increased in critically ill children with acute kidney injury (median 355 ng/mL, IQR 166–1322 ng/mL) compared with those without acute kidney injury (median 186 ng/mL, IQR 98–365 ng/mL; p = .009).Serum NGAL is a highly sensitive but nonspecific predictor of acute kidney injury in critically ill children with septic shock. Further validation of serum NGAL as a biomarker of acute kidney injury in this population is warranted.